Cell adhesion through clustered ligand on fluid supported lipid bilayers

Article abstract of DOI:10.1039/b924523e

The quartz crystal microbalance technique with dissipation monitoring and the complementary optical microscopy technique were used for monitoring cell adhesion on an RGD-functionalized supported lipid bilayer. A critical interligand RGD spacing of nearly 80 nm was estimated for cell adhesion when an RGD spacing of 10 nm was necessary to observe flattened cells on a fluid surface.

Full text of DOI:10.1039/b924523e

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Cell adhesion through clustered ligand on fluid supported lipid bilayers†
Ludivine Sandrin, Liliane Coche-Gu e´ rente, Amandine Bernstein, Hajra Basit, Pierre Labb e´ , Pascal Dumy* and
Didier Boturyn*
Received 25th November 2009, Accepted 1st February 2010
First published as an Advance Article on the web 12th February 2010
DOI: 10.1039/b924523e
The quartz crystal microbalance technique with dissipation
monitoring and the complementary optical microscopy tech-
nique were used for monitoring cell adhesion on an RGD-
functionalized supported lipid bilayer. A critical interligand
RGD spacing of nearly 80 nm was estimated for cell adhesion
when an RGD spacing of 10 nm was necessary to observe
flattened cells on a fluid surface.
Cell-surface recognition is a typical event in biological systems that
triggers a wide range of cellular processes such as cell adhesion,
growth, differentiation, migration and survival. Among the pro-
tagonists of these processes, the integrins constitute an important
family of transmembrane receptors that bind to the proteins
1
Fig. 1 Structure of clustered RGD-containing compounds.
of the extracellular matrix (ECM). The RGD (Arg-Gly-Asp)
tripeptide was identified as a minimal peptide sequence for cell
2
adhesion in many ECM proteins. Consequently, various materials
Very recently, we measured the constant of affinity (K
clustered RGD-containing compound for purified integrins.
D
) of this
have been functionalized with RGD-containing peptides for a
wide range of applications including drug discovery, biosensors
13
K
D
3
values rose from 3.9 nM for the multimeric RGD-containing
compound to 41.7 nM for cyclo[-RGDfK-]. To further explore the
adhesive properties of the clustered RGD-containing ligand, we
herein report on studies of RGD-mediated cell adhesion to an SLB
surface. We provide for the first time the average ligand spacing
to trigger cell adhesion and spreading on a fluid substrate. To
this end, we used the quartz crystal microbalance with dissipation
monitoring (QCM-D) in combination with optical microscopy.
We then prepared compound 1 which incorporates a lipid
surface anchoring domain. Scheme 1 gives an overview of the route
to construct lipid conjugate 1. We preferred the oxime bond forma-
tion to link aldehyde-bearing RGD motifs to the peptide scaffold
displaying four aminooxy functions. This reaction proved to be
and tissue engineering. For instance, binding molecules such as
cyclo(-RGDfK-) pentapeptide have shown strong selectivity to the
4
a
V
b
3
integrin, the vitronectin receptor up-regulated on endothelia
during tumour angiogenesis. Very recently, this peptide was further
exploited to study cell adhesion on a passivated glass surface
5
through clustered RGD-containing gold nanoparticles. Experi-
mental results reveal that cell adhesion occurs exclusively when
the average ligand spacing is smaller than 70 nm. Alternatively,
6–10
studies on fluid lipid bilayers were reported in the literature.
Peptide ligands are then immobilized in the form of amphiphilic
lipopeptides. The supported lipid bilayers (SLBs) are suitable fluid
systems from which to examine cell receptor–ligand interactions
in a biologically relevant environment. Moreover, they enable
a precise control of ligand concentration and presentation, and
allow their lateral mobility.
14
highly efficient and chemoselective. Cyclopeptides were prepared
through a combination of solid and solution-phase syntheses,
15
according to methods previously described. Building blocks 2
and 3 encompassing respectively the protected aminooxy function
and the palmitoyl group were introduced during the solid phase
peptide synthesis (SPPS). This strategy reduces the number of
steps and the combination of protecting groups required so far for
the construction of such lipopeptides. We used 1-ethoxyethylidene
group (Eei) to protect the aminooxy function since we found that
this group is entirely compatible with the SPPS. Briefly, linear
lipopeptide 4 was obtained using standard Fmoc/t-Bu solid-phase
chemistry on an acid-labile Rink amide resin. The subsequent
head-to-tail cyclization was achieved after the removal of the allyl
group affording the expected lipopeptide 5 (Scheme 1). Aqueous
trifluoroacetic acid was used for the deprotection of the aminooxy
functions and the release of the cyclopeptide from its support.
Finally, compound 1 was easily obtained from the oxime ligation
of the appropriate cyclopentapeptide cyclo[-RGDfK(-COCHO)-].
We previously described a clustered RGD-containing ligand
that exhibits attractive biological properties for the imaging
11
12
of tumours
and for targeted drug delivery (Fig. 1). This
compound is based on a cyclic decapeptide scaffold that presents
in a spatially controlled manner two independent functional
domains: a clustered ligand domain for integrin recognition
conferring cell targeting and a domain devoted to a supplementary
function (cancer monitoring, drug delivery, anchoring device).
16
D e´ partement de Chimie Mol e´ culaire, UMR CNRS/UJF 5250, ICMG FR
2
607, 301 rue de la chimie, BP53, 38041 Grenoble cedex 9, France. E-mail:
didier.boturyn@ujf-grenoble.fr; Fax: (+33) 4 76 51 49 46; Tel: (+33) 4 76
5
1 45 23
†
Electronic supplementary information (ESI) available: Details of the
peptide syntheses, syntheses and spectroscopic characterisation of com-
pound 1, SLB formation, cell culture, QCM-D, AFM and microscopy
experiments, optical images of cell adhesion. See DOI: 10.1039/b924523e
This journal is © The Royal Society of Chemistry 2010
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Scheme 1 Synthesis of compound 1. a) Fmoc-aa-OH (2 equiv.), PyBOP (2 equiv.), DIPEA (3–4 equiv.), 30 min; b) piperidine–DMF (1 : 4); c) Pd(PPh
0.2 equiv.), PhSiH (100 equiv.), CH Cl , 30 min then PyAOP (2 equiv.), DIPEA (4 equiv.), 30 min; d) TFA–H O–triisopropylsilane (90 : 5 : 5), 30 min;
e) cyclo[-Arg-Gly-Asp-D Phe-Lys(-COCHO)-] (4.4 equiv.), CH CN–H O (1 : 1).
3 4
)
(
3
2
2
2
3
2
Supported lipid bilayers were then prepared by small unil-
amellar vesicle fusion to a silica sensor surface. We used neu-
tral phospholipids such as palmitoyloleoyl phosphatidylcholine
cell adhesion at different RGD-displaying surfaces. A decrease
in frequency indicates an increase in coupled mass to quartz
crystal, while an increase in dissipation is caused by damping of
the crystals oscillation, indicative of non-rigid viscoelastic nature
of the cell. Additionally, QCM-D does not sense the mass increase
from cells that simply settle on the sensor and loosely attach to the
surface by unspecific interactions. This technique is only sensitive
to adherent cells that interact with the substrate through integrin
receptor. As a consequence, QCM-D technique was blind to HEK-
293(b3) cells interacting with pure POPC surface or when HEK-
293(b1) cells were applied to the surface displaying 1% molecule
1 (Fig. 2). This result indicates that our SLB do not produce non-
(
POPC) in combination with controlled amount of lipopeptide 1
to prevent non-specific cell adhesion. The assembly of the surface
was monitored by QCM-D experiments and characterized by
means of an atomic force microscope (AFM) (see Supplementary
Information). AFM images do not reveal lipopeptide aggregation
but a homogenous dispersion of compound 1 within SLB. To
assess the cell adhesion studies, we prepared SLBs encompassing
increasing clustered ligand densities (molar ratios from 0.01%
to 5%). In this work, the size of RGD ligand 1 is nearly 3 nm
in diameter. Since the a
v
b
3
integrin diameter is approximately
specific cell adhesion. When the a
v
b -expressing cells were applied
3
17
1
0 nm, each molecule 1 is able to bind strictly one integrin.
to the different RGD-presenting surfaces, the frequency and the
dissipation shifts increased gradually for loadings greater than
0.01% ligand 1 (Fig. 2). This signifies that cell adhesion is triggered
by the RGD motif and that the increase of the mole fraction
of compound 1 leads to an increase in the number of adhered
cells in close contact with the resonator surface. At the limit of
0.01% ligand 1 (red lines), we did not detect HEK-293(b3) cell
adhesion using our experimental conditions. At this ligand density,
The capacity of the surface for selective cell binding through
RGD-integrin recognition was then evaluated by QCM-D in
combination with optical microscopy. The QCM-D technique is
18
attractive as it allows in situ real-time measurements.
Experiments were conducted using human embryonic kidney
cells HEK-293(b3) that overexpress a integrin and HEK-
93(b1) (a negative but expressing a ) chosen as the negative
v
b
1
3
2
v
b
3
v
b
2
control cell line. Moreover, the time of cell experiments was limited
considering the average area of 0.7 nm per phospholipid in the
20
to 1 h since cells are known to secrete and bind to their own ECM
fluid state, we estimate the ligand densities of approximately 140
19
-2
components. Fig. 2 depicts the QCM-D profiles recorded during
ligands mm and the critical interligand spacing of nearly 84 nm
21
for cell adhesion. Very recently, developments in nanotechnology
have given access to the determination of ligand spacing on a
passivated glass surface: cell adhesion is strongly diminished when
5
RGD motif spacing is higher than 70 nm. Besides, the integrin
expression level was determined to be nearly 10 proteins/cell,
yielding an integrin density of 100 proteins mm for spread cells.
5
-
2
22
Together, these data are in good agreement with our results and
suggest that all integrin receptors need to be engaged for cell
binding on a fluid supported lipid bilayer.
To further discriminate between round cells and flattened
cells, during QCM-D experiments we simultaneous observed cell
morphology by using a camera system (Fig. 3, see also Supplemen-
tary Information). On a surface displaying 1% ligand 1 (14 300
-
2
ligands mm , interligand spacing of ~ 10 nm), the HEK cells
assumed a flattened morphology (~ 40 mm in diameter) with some
filopodial extensions. Moreover, cells are able to move on the fluid
surface (Fig. 3 A–C). These results involve the formation of actin
stress fibers and suggests that the RGD motifs on the lipid bilayer
membrane induce integrin-mediated tyrosine phosphorylation as
Fig. 2 Evaluation of cell adhesion using QCM-D experiments. Cell
-1
-1
suspension (100 000 cells mL ) was continuously injected at 100 mL min .
Resonance frequency shift (solid lines) and energy dissipation shift (dashed
lines) for HEK-293(b3) applied to 5% (green), 1% (light blue), 0.1% (black),
0
.01% (red), w/o ligand 1 (purple), and for HEK-293(b1) applied to 1%
23
ligand 1 (pink).
described for cells plated on ECM protein-coated surfaces. It
1
532 | Org. Biomol. Chem., 2010, 8, 1531–1534
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suggest that SLB containing 1% RGD ligand 1 could potentially
be used as a biomimetic membrane, and we envisage exploiting
this model system in evaluating the cellular response to different
lipid bilayer membranes of varying composition.
Acknowledgements
This work was supported by the Institut National du Cancer
(
INCA), the Universit e´ Joseph Fourier, the Centre National de
la Recherche Scientifique (CNRS), the Nanoscience Foundation
and NanoBio (Grenoble). We gratefully acknowledge Dr J.-C. Coll
(
Institut Albert Bonniot, INSERM U823, La Tronche, France) for
providing us with human embryonic kidney cell lines.
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